Among photosynthetic microorganisms concerned by the invention are more particularly represented aquatic plants such as for example micro-algae, moss protonemas, small macro-algae and isolated cells of multicellular plants. These aquatic plants have interesting properties in the fields, notably of pharmacy, human and animal nutrition, of dermo-cosmetology, of energy and of the environment.
Like for most photosynthetic microorganisms, access to this resource essentially consists in assisted culture in suitable reactors. As light is their main substrate, the culture medium should have an optical interface receiving a light flux. The difficulty of cultivating photosynthetic microorganisms lies in the fact that they themselves form obstacles to the passage of light, which is their main substrate. The growth of the culture will therefore stabilize when light no longer penetrates into the thickness of the culture. This phenomenon is called self-shadowing.
The length of the optical path or “light path length”, allows characterization of the different confinement modes and is defined as being:
the length of the light path from its entry into the culture through a transparent optical interface to as far as an opposite opaque wall; or
the half of the distance separating both transparent optical interfaces when the confinement receives light through two opposite transparent optical interfaces.
This optical path length varies between a few centimeters and a few decimeters and essentially determines the propipeion of biomass per unit time and per optical unit surface (surface propipeivity in g/m2/d) and the concentration of the culture between (in g/L) in the final growth phase. The various confinement modes which are applied for ensuring the culture of small aquatic plants may thus be classified according to this characteristic length.
The photosynthesis reaction is also accompanied by consumption of carbon dioxide (CO2) and by propipeion of oxygen (O2). Excess oxygen inhibits the reaction, while the absence of carbon dioxide interrupts it through lack of substrate to be transformed. A gas/liquid interface should therefore be placed for mass transfers between these gases and the liquid phase. In order to promote these exchanges and to avoid heterogeneities, the culture should be the centre of a mixture intended to renew the organisms at the aforementioned optical interface and also at this gas/liquid interface.
A first known embodiment of a photosynthetic reactor consists in an open container of the basin or tank type where the culture is maintained by gravity and has a free surface achieving by itself the optical interface and the liquid/gas interface. The culture is mixed inside the basin by one or more mechanical stirring devices, for example of the paddle wheel type. The thereby achieved basin cultures may cover significant surface areas and this embodiment is at the origin of the essential of the present world propipeion of micro-algae, which attains several thousand metric tons of dry weight. Photosynthetic organisms produced by this type of reactor are essentially:
so-called extremophilic algae, the media of which are hostile to predators and competitors, such as for example algae of the spirulin or Dunaliella type; or
so-called dominating algae, which withstand mechanical stresses or contaminations better than the other ones, such as for example the algae of the Chlorella, Scenedesmus, Skeletonema, Odontella or Nannochloropsis type.
A second known embodiment of a photosynthetic reactor also consists in an open container of the reservoir or tank type, but the dimensions of which are smaller than that of the basins of the first known embodiment. These containers generally have lateral walls transparent to solar radiation, so that the optical interface is formed both by the free surface of the liquid medium and by the transparent lateral walls.
In this second embodiment, it is conventional to resort to injection of air carried out in the low portion of the reservoir which leads to the formation of air bubbles moving up in the liquid to the free surface. The surface of the thereby formed bubbles is the gas/liquid interface. By moving up to the surface, the bubbles carry away the culture upwards, thereby generating convective motions which may extend to the whole volume. Carbon dioxide (CO2) is sometimes added to the injected air in order to provide extra carbon according to a predefined molar fraction of a few percent.
With a volume of less than that of the basins of the first embodiment, the reservoirs of the second known embodiment are adapted to more controlled cultures, in particular to the cultures of micro-algae intended for feeding larvae of molluscs or living preys of fish larvae in aquaculture. Frequent cleaning of these reservoirs as well as pure and bulk inoculations allow limitation of the contaminations inside the reservoir. With a number of several tens of species, the thereby cultivated micro-algae have temperature and light needs relatively close which makes their culture possible in premises common thereto.
Both of these embodiments in the form of an open container provide an optical path length from one to several decimeters.
A third known embodiment of a photosynthetic reactor consists in a closed reactor, a so-called photobioreactor and which comprises a closed mouth inside which the liquid culture medium circulates, said closed loop comprising a reaction pipe provided with reaction sections made in a material transparent to the light radiation (or to light), and a return pipe ensuring the connection between both opposite ends of the reaction pipe.
The photobioreactors, notably described in documents GB 2,118,572 A, ES 2 193 860 A1, GB 2,331,762 A, ES 2 150 389 A1, FR 2 685 344 A1 and FR 2 875 511 A3, provide substantially smaller optical path lengths, of the order of one to several centimeters, as compared with the embodiments with an open container, and they give the possibility of attaining concentrations of photosynthetic organisms of several g/L sheltered from aerial contaminations. The reaction pipe of photobioreactors generally consists in transparent plates or tubes, in glass or plastic, with a thickness and a diameter of the order of 1 cm, which are connected end to end through bends in order to form together a pipe in the form of a coil.
The return pipe comprises a so-called ascending vertical tube in which the liquid medium moves upwards, and a descending vertical tube in which the liquid medium moves down under the effect of gravity.
The gas injection system generally applied in photobioreactors consists in a gas siphon, otherwise called a “gas-lift” or a gas lifting device, i.e. in an injection of gas at the base of the ascending vertical tube of the return pipe; said gas injection being used both for circulating or displacing the liquid reaction medium and for achieving gas-liquid exchanges. The gas lift includes in its high portion a reservoir with a load or widened volume wherein the lower circulation rates allow gas-liquid separation, and the descending vertical tube of the return pipe opens out in the bottom of the load reservoir for feeding the reaction pipe with liquid.
The aforementioned photobioreactors apply the principle according to which the reaction only occurs in the liquid phase, in other words these photobioreactors seek to minimize the volume of injected gas into the reactor so as not to reduce the volume of the liquid culture medium by as much, with the concern of not reducing propipeion. Thus, in these photobioreactors, extraction of oxygen is achieved by means of the vertical ascending tube defined above; said vertical ascending tube forming a column of air bubbles opening out into the load reservoir receiving the liquid culture medium, and including injection of gas in the low portion, opportunely CO2-enriched air. As described above, both circulation and gas transfer functions coincide within this single device called a gas lift, which generates an ascending vertical circulation by exchange of momentum between the liquid mass and the gas bubbles resulting from the injection. The photosynthetic oxygen in oversaturation in the liquid passes into the gas phase by sweeping with air, while CO2 passes into solution. These degassing and carbonation functions are indispensable and occur at this single device simultaneously.
The gas lifts have the drawback of generating gas bubbles which move up the vertical ascending tube of the return pipe of the photobioreactors. The applicant has actually observed the deleterious role of these bubbles for cultivating microorganisms in photobioreactors:
the bubbles mechanically stress the micro-algae and may be detrimental to fragile microorganisms on the one hand; and
the bubbles capture by a surfactant effect the molecules which have surfactant properties, and notably organic molecules, cell debris and excretion propipes of living cells on the other hand.
These substances normally dispersed in the medium in the absence of bubbles are thus gathered as aggregates at the free surface of the load reservoir when the bubbles burst. The bacteria and fungi which could not develop because of the strong dilution of these organic molecules then find concentrated substrates favorable to their development.
One of the goals of the present invention is to avoid or at least limit the formation of bubbles in order to:
contain the bacterial and fungal development for example in order to remain compatible with conventionally imposed sanitary standards in the culture of microorganisms; and
limit the mechanical stresses in the liquid culture medium, and thereby allow the cultivation of certain fragile microorganisms which were up to now excluded from such cultivation in a reactor.
In an alternative embodiment of the gas lift, deoxygenation of the liquid culture medium circulating in the photobioreactor is obtained by causing the liquid medium to gravitationally fall into a container with a constant level. The liquid culture medium is here circulated by a pumping means, notably of the centrifugal pump type, positioned in the reaction pipe designed for not only compensating for the pressure losses in the pipe but also for raising the culture by the height of the fall.
Although it generates less bubbles, this device with a centrifugal pump is also mechanically damageable for the microorganisms and for the gas lift. Indeed, in order to overcome the pressure losses, there is generation, at each passage at right angles to the pumping means, of mechanical forces which may impede the growth of the microorganisms and cause mortalities within the culture. The propipeion performances are then found to be altered, sometimes in a redhibitory way.
For example, it was seen that it is not possible to cultivate certain so-called fragile micro-algae in photobioreactors including centrifugal pumps for circulating the culture. These fragile micro-algae seem to be all the more sensitive to mechanical stresses since they form chains and/or they have appendices such as bristles, flagella, and spicules. Certain micro-algae, such as for example the algae of the Haematococcus pluvialis type, lose their flagella and encyst by forming a thick and resistant cell wall. On the other hand, other micro-algae such as for example the algae of the Chlorella vulgaris or Nannochloropsis oculata kind, do not have any appendage and have a thick cell wall, so that the latter resist to passing into the pumping means and notably into the centrifugal pumps.
However, it is difficult to identify the nature of the mechanical stresses having an influence on the survival and growth of the microorganisms. Most authors agree for stating that shearings and accelerations have the most influence. Shearings generate tensions which may alter the cell integrity with tearing of the wall of the microorganisms and effusion of the cytosol. Accelerations alter the structure of the cell by increasing the gravitational field.
Living cells are poorly prepared for these forces, and perhaps even more, aquatic cells which live in hydrostatic equilibrium and which have not developed any structure capable of overcoming a gravitational field. Furthermore, aquatic cells are sensitive to threshold values and also probably to the changes and to the duration of exposure. In the present state of knowledge, it is difficult to predict the mechanical effects of hydrodynamic conditions imposed to the cells.
One of the objects of the present invention is to reduce the mechanical effects imposed to the microorganisms, notably the effects of the shearing and acceleration type, in order to extend the number of cultivable species inside the reactor to those which are the most sensitive to these damageable mechanical effects, in other words provide a reactor allowing the cultivation of fragile microorganisms, such as for example the fragile micro-algae mentioned above.
Further, the applicant observed that the culture yield of photobioreactors equipped with a gas lift or a centrifugal pump was limited notably because of the formation of bubbles. Indeed, the applicant established that the culture yield partly depends on phenomena involved in the gas-liquid transfer in order to avoid losses and reduce this significant item of expenditure. Modeling the gas-liquid transfer of carbon dioxide intended for the reaction and of oxygen which it produces, requires determination of the transfer rate which depends on the surface transfer coefficient.
The surface transfer coefficient is a key parameter which expresses the performances of a gas/liquid exchange system in the stable condition. This surface transfer coefficient is equal to the propipe of the bulk transfer coefficient of material towards the liquid “KL” (m·s−1) and of the interface area reduced to the volume “a” (m−1), wherein:a=(αG·S)/V a: Interface area reduced to the volume (m−1);αG: Phase retention coefficient;S: Contact surface area (m2); andV: Volume of the reactor (m3).
The surface transfer coefficient therefore depends on the geometry of the gas/liquid exchange system but also on the physicochemical properties of the liquid and of the gas. In the case of a gas/liquid exchange within a vertical bubble column, the exchange surface area depends on the number of bubbles and on their size. The population of bubbles generated by gas injection in a liquid depends on the injection flow rate, on geometry of the injector, and on the pressure difference on either side of the latter.
The present invention notably has the goal of providing a photosynthetic reactor which allows bulk cultivation of photosynthetic microorganisms and its extension to the most fragile species, with a reactor which meets the following problems:
reduce or even avoid mechanical stresses generally related to stirring and to circulation of the culture medium and which reduce the survival and growth performances of photosynthetic microorganisms such as micro-algae and more particularly micro-algae in chains provided with appendages;
reduce or even avoid the propipeion of bubbles of small dimensions which may promote aggregation of organic molecules and the development of heterotrophic microorganisms for which they are used as a substrate;                while achieving photon transfer, in order to deliver the solar radiation to the photosynthetic microorganisms, the mass transfer or the gas/liquid transfer indispensable for providing the carbon and removing the oxygen, and the thermal transfer, in order to remove the calories brought by the radiation and maintain the culture at the right temperature; and        
while maintaining mechanical conditions preserving the integrity of the cells and avoiding the exchanges with the surrounding medium lending themselves to contaminations and disseminations.